This invention relates to a protein having the activity for vacuolar compartmentalization of flavonoids in plant cells, as well as a gene encoding the protein. The invention also relates to a transformed plant harboring the gene.
Flavonoids are the secondary metabolites unique in the plant kingdom. They include three major subclasses of compounds: flavonols, anthocyanins, and proanthocyanidins (PAs; so-called condensed tannins). Despite the multitude of functions of flavonoids in plants such as UV-B protectants, signaling molecules between plants and microbes, and regulators of auxin transport (reviewed in Winkel-Shirley, B. (2001) Plant Physiol. 126, 485-493), loss or deficiency in flavonoids has generally no deleterious effect on plant growth and development, and is easily detected as a change of color in some specific organs. These facts prompted the present inventors to isolate mutants with reduced or varied coloration in order to uncover the flavonoid biosynthetic pathway in plants.
Changes in flavonoid pigments in maize kernels are one of the topics most intensively studied so far, which contributed to the establishment of anthocyanin pathway. Given the purpose of molecular breeding in ornamental plant species, a number of mutants have been isolated in petunia and snapdragon (Mol, J., Grotewold, E., and Koes, R. (1998) Trends Plant Sci. 3, 212-217). Over the last decade molecular genetics in Arabidopsis has been developed. Most Arabidopsis mutants deficient in flavonoid pigments have been described as transparent testa (tt) (Koornneef, M. (1990) Arabidopsis Inf. Serv. 27, 1-4).
To date, 21 tt loci have been identified, and about a half of them have been analyzed in detail. Analysis on the tt mutants achieved cloning and characterization of a number of structural and regulatory genes in Arabidopsis flavonoid pathway (FIG. 1). Because the structural genes are single-copy except for flavonol synthase (FLS), the Arabidopsis flavonoid biosynthetic pathway is valuable as a model to analyze regulation and subcellular organization for plant metabolisms (reviewed in Winkel-Shirley, B. (1999) Physiol. Plant. 107, 142-149).
The flavonoid synthesis proceeds in the cytosol, whereas most of their endproducts are finally accumulated in the vacuoles. Because many secondary metabolites including flavonoids are cytotoxic and genotoxic even in the cells that produce them, it is thought that there is a sequestration system that is analogous or related to that for exogenous toxic compounds in plants. Detoxification of xenobiotics in plants is composed of three phases: (I) activation phase which usually involves hydrolysis or oxidation to realize higher reactivity, (II) conjugation phase of compounds metabolized in phase I with hydrophilic molecules such as glucose, malonate or glutathione, and (III) export phase from the cytosol by membrane-associated transport proteins (Coleman, J. O. D., et al (1997) Trends Plant Sci. 2, 144-151).
Major reaction in phase I is catalyzed by the cytochrome P-450, and some P-450 enzymes are involved in the flavonoid biosynthetic pathway such as cinnamate 4-hydroxylase, F3′H, F3′5′H (Winkel-Shirley, 2001, supra). With respect to detoxification of anthocyanins, conjugation with glucosyl moieties at 3 position is necessary to solubilize the precursors (anthocyanidins), and it is said that the corresponding transferase, UDP-glucose:flavonoid glucosyltransferase (UFGT), is one of the structural enzymes in anthocyanin pathway. Based on the structures of anthocyanins identified to date, they must undergo various modifications such as methylation, acylation, and glycosylation, and some corresponding genes have been identified in petunia (e.g., Brugliera, F., et al. (1994) Plant J. 5, 81-92).
In addition, it was reported that glutathione S-transferase (GST) is essential for anthocyanin pigmentation. Maize BZ2 and petunia AN9 encode GST proteins, and they can functionally complement each other (Alfenito, M. R., et al., (1998) Plant Cell 10, 1135-1149). The function of these GSTs was firstly thought to be the one of forming glutathione-conjugates of anthocyanidin-3-glucosides (Marrs, K. A., et al. (1995) Nature 375, 397-400).
In comparison with anthocyanins, modification and compartmentalization of PAs or their precursors are more poorly understood. The current hypothetical model for PA accumulation mechanisms depends largely on the data from Douglas fir (reviewed in Stafford, H. A. (1989). The enzymology of proanthocyanidin biosynthesis. In Chemistry and significance of condensed tannins (Hemingway, R. W. and Karchesy J. J. eds). New York: Plenum Press, pp. 47-70.).
It has been believed that PAs are composed of flavan 3-ols and flavan 3,4-diols (leucoanthocyanidins), the former of which as start units and the latter as extension units, but another pathway involving 2,3-cis-flavan 3-ols as extension units was recently suggested (Xie, D.-Y., et al. (2003) Science 299, 396-399: FIG. 1).
It is likely that their uptake into the vacuoles (or the lumen of the endoplasmic reticulum; Stafford, 1989, supra) is performed as monomer forms but not as polymer forms (Debeaujon, I., et al. (2001) Plant Cell 13, 853-871). The precursors are progressively condensed and the polymers formed are oxidized, resulting in brown coloration (FIG. 1).
The condensation and oxidation steps are probably performed enzymatically, while non-enzymatic reactions can be easily done (Stafford, 1989, supra). Some barley mutants presumably involved in condensing and/or accumulation steps were reported as tannnin (proanthocyanidin)-deficient (ant) mutants (Gruber, M. Y., et al., (1999) Genetic systems for condensed tannin biotechnology. In Plant Polyphenols 2: Chemistry and Biology. (Gross, G. G., Hemingway, R. W., and Yoshida, T. eds). New York: Kluwer Academic/Plenum Publishers, pp. 315-341), but molecular and biochemical evidence for their compartmentalization, polymerization and oxidation after the synthesis of PA precursors has to be awaited.
In Arabidopsis, compartmentalization mechanisms for flavonoids, even for anthocyanins, remain to be clarified, as compared with their biosynthetic pathway (FIG. 1). This situation is accounted for mainly by the fact that most of tt mutants are restricted to those which are defective in flavonoid ‘synthetic’ steps but not in ‘transport’ steps. The exception is the case of tt12 mutant. Debeaujon et al. (2001. supra) have isolated TT12 gene and suggested that TT12 is a putative transporter, which is responsible, at least in part, for vacuolar sequestration of PA precursors in Arabidopsis seed coat.
The present inventors previously obtained two novel tt mutants during investigation of mutation rate of ion beam irradiation in Arabidopsis (Shikazono, N., et al. (2003) Genetics 163, 1449-1455). One is a tt18 mutant (formerly named as tt19 in Winkel-Shirley, 2001, supra), in which a gene encoding a putative leucoanthocyanidin dioxygenase (LDOX) is impaired. The other is defined as a tt19 mutant, but to date, neither the causative gene nor the nature of the tt19 mutant has been elucidated.